The study included three blue grape varieties of Vitis vinifera L.  and four white grape varieties . From the viewpoint of producing extracts with high stilbenes content, the Hibernal variety is clearly the best. The mean amounts of the stilbenes for this variety at all localities and for all three years were 4.99 g/kg for trans-resveratrol, 3.24 g/kg for trans-ε-viniferin, and 1.73 g/kg for r2-viniferin. The influence of vintage, locality, and variety on the amounts of stilbenes was studied using PCA analysis. In contrast to expectations, there was no strong impact of locality on stilbenes content. The differences were varietal for most varieties, regardless of the area of cultivation. Laurot and Hibernal varieties did differ significantly in that respect, however, as they exhibited clear dependence on location.Grape canes are a waste product from viticulture that show potential as an industrially extractable source of stilbenes, which are valuable for medical and other purposes. In this work, grape canes collected in three consecutive years (2014–2016) at six different places in South Moravia, Czech Republic were extracted, and the contents of  Considering the large amounts of stilbenes found in grape canes (3.45 g of trans-resveratrol per kg dry weight (d.w.) of cane and 1.3 g of trans-ε-viniferin per kg d.w. of cane) and their market values, it is estimated , Vitis riparia, Vitis rupestris, Vitis vinifera, Muscadinia rotundifolia, Vitis vinifera x Muscadinia rotundifolia). Those authors isolated 15 stilbenoids, confirmed their structures by mass spectrometry and nuclear magnetic resonance spectrometry, and found that the greatest yield of trans-ε-viniferin was 5.74 g/kg in variety Vitis riparia Pull. Vergara et al. [trans-resveratrol, trans-piceatannol, and trans-ε-viniferin in Chilean Pinot Noir and Gewürztraminer and found the highest concentration of trans-ε-viniferin of 0.87 g/kg in canes of Pinot Noir. They also found in this variety that—in contrast to other varieties—it is advantageous to keep the canes after trimming in the vineyard for a period of two months, during which the content of the trans-ε-viniferin practically doubled.Pawlus et al.  studied 2O mixture at room temperature. The most abundant stilbene in all cultivars was trans-ε-viniferin, which reached concentrations ranging from 0.90 g/kg d.w. in Palomino fino to 2.81 g/kg d.w. in Gewürztraminer. Those authors also used principal component analysis (PCA) to better understand the differences between cultivars.Guerrero et al.  describetrans-resveratrol in the past and in trans-ε-viniferin today is due to their biological properties. Certain of these properties are very promising from the viewpoint of human medicine. For example, trans-ε-viniferin glucoside is an attractive new candidate for protecting brain cells, and could therefore be useful in treating Alzheimer’s disease [trans-(−)-ε-viniferin activity increases the activity of mitochondrial sirtuin 3 and protects cells in a model of Huntington’s disease, which is virtually untreatable [Trans-ε-viniferin is active not only as a preventive agent against cancer, but also as a substance having direct cytotoxicity against selected cancer cell lines [trans-ε-viniferin and vancomycin is active against methicillin-resistant Staphylococcus aureus—a bacterium responsible for difficult infections in humans and animals. Stilbenes from canes have also been studied as natural fungicides. Methanolic and ethanolic crude extracts from grape canes of Pinot Noir, Gamaret, and Divico varieties have exhibited significant antifungal activity against three major fungal pathogens affecting grapevines: Plasmopara viticola, Erysiphe necator, and Botrytis cinerea [Vitis vinifera L. wastes  have recently been used as a cheap source of bioactive compounds for the development of natural fungicides against, for example, Plasmopara viticola [trans-ε-viniferin in far greater amounts, a successful search for sources of these substances and a description of their distribution in different grapevine varieties with regards to sampling procedure and storage conditions is highly desirable and promising. The great interest in  disease . This prreatable . Trans-εll lines . The resll lines  are very cinerea . Stilbenviticola . BecauseAs is apparent from the overview presented above, it is necessary to thoroughly study the distribution of stilbenes in canes in relation not only to the variety and sampling points, but also to the variability in stilbenes content through time. Our work was therefore focused on studying and comparing stilbenes contents of seven varieties of grape canes in six different places in South Moravia, Czech Republic during a three-year study. To our knowledge, this is the first such comprehensive study of its kind.Vitis vinifera L. from the Moravian wine region were included in the experiment, encompassing three blue grape varieties: Cabernet Moravia , Blaufränkisch , and Laurot ; and four white grape varieties: Chardonnay , Green Veltliner , Hibernal , and Malverina . The varieties and six sampling sites are described in the Materials and Methods. In our study during 2014–2016, we focused on determining three dominant stilbenes in grape canes: trans-resveratrol, trans-ε-viniferin, and r2-viniferin.Seven varieties of trans-resveratrol were found in the Blaufränkisch (Bl) variety at all three sampling points and during all three observed years. The lowest values were recorded in 2014, and the difference was statistically significant as compared to 2015 and 2016  was always recorded at Sampling Site 6 when comparing the same variety. With the exception of the Hibernal variety, the lowest value among different varieties in 2016 was also recorded at Sampling Site 6. Findings in the year 2015 were very diverse.Regarding r2-viniferin, it is interesting that in 2014, the lowest value . Hi-6 differed from the other Hibernal sites. The other varieties exhibited practically no dependence on the area of cultivation , but a dependence on variety was obvious in the case of Chardonnay  vs Cabernet Moravia  vs Blaufränkisch . The variance in values for Green Veltliner and Malverina varieties was slightly larger.In 2016, the Hibernal variety differed considerably from the others, and Sampling Sites 6 and 4 for Hibernal differed from Sampling Site 1.The present results indicate that the difference is varietal for most varieties, regardless of the area of cultivation (sampling sites). Laurot and Hibernal varieties differ significantly, as in their cases we found a clear dependence on location (sampling sites).The above results are complemented by PCA including all three years . For theThe issue of the diversity of the distribution of stilbenes obtained from grapevine canes is rather complex. As we have already pointed out in the article, the processes themselves (different storage and drying times) affect the contents of these substances. Our previous article  studied trans-resveratrol), the higher rainfall in 2014 might have increased the fungal pressure. This means that plants might have been stressed, and in this way the content of trans-resveratrol increased. It is obvious from From the studied parameters, rainfall resulted higher in 2014. Due to the stress conditions which induce stilbene biosynthesis  , but the effect was not found to be the same for all varieties. The reason may be that the varieties are not grown evenly across South Moravia. For all investigated varieties, the dominant stilbenes were Vitis vinifera L. at various locations has not yet been published in the literature.To our knowledge, such an extensive set of data from a three-year study about the content of stilbenes in canes of different varieties of Vitis vinifera L. canes from the Moravian wine region were included in the experiment—three blue grape varieties: Vitis vinifera L. variety Cabernet Moravia (CM), Vitis vinifera variety Blaufränkisch (Bl), and Piwi variety Laurot (La); and four white grape varieties: Vitis vinifera L. variety Chardonnay (Ch), Vitis vinifera L. variety Green Veltliner (GV), Piwi variety Hibernal (Hi), and Piwi variety Malverina (Ma). The six sampling sites (1–6) are described in Seven varieties of Collected canes were dried in darkness for three months at room temperature. They were then ground using a laboratory grinder. Powdered grape canes were extracted for 165 min at 50 °C as described in our previous publication [trans-resveratrol was calculated according to a calibration curve for trans-resveratrol. The content of trans-ε-viniferin was calculated according to the calibration curve for trans-ε-viniferin, the content of r2-viniferin was calculated according to the calibration curve for trans-ε-viniferin in and the obtained value was multiplied by the ratio of MW of r2-viniferin/MW of trans-ε-viniferin. Data were recorded as mg/kg dry weight. The method was validated in terms of limits of detection and of linearity [Trans-resveratrol , trans-ε-viniferin . The samples were separated using an HP 1050 high-performance liquid chromatography instrument  on a 3 µm, 150 mm × 2 mm, Luna C18(2) column  with water-acetonitrile-o-phosphoric acid mobile phase. We used a G1315B diode array detector  and G1321A fluorescence detector . We followed the method detailed in our previous publication [inearity . Trans-rtrans-resveratrol (purity > 99%) from Sigma-Aldrich , trans-epsilon viniferin from Sigma-Aldrich , methanol, and acetonitrile from Merck , and ortho-phosphoric acid p.a. from Fluka . Standards were purchased from the following sources: trans-resveratrol, trans-ε-viniferin, and r2-viniferin in studied cane extracts from six different places in South Moravia and from seven varieties of Vitis vinifera L. did not show the expected strong impact of locality. The largest difference was found in the variety Piwi Laurot at Sampling Site 2 for trans-resveratrol and r2-viniferin. A significant difference was also observed in the Cabernet Moravia variety at Sampling Sites 3 and 6 for r2-viniferin. From the viewpoint of producing extracts with high stilbenes content, the Hibernal variety is clearly the best. The mean amounts of the aforementioned stilbenes for this variety at all localities and for all three years was 4.99 g/kg for trans-resveratrol, 3.24 g/kg for trans-ε-viniferin, and 1.73 g/kg for r2-viniferin. The measurements of


For the first time, a hard wear-resistant multi-layer of TiCrN-TiAlN-TiAlSiN-TiAlSiCN was deposited on carbon steel CK45-based tillage tools to increase their useful lifetime. The layers were deposited by using an arc-PVD method without post-annealing procedures. XRD and EDX data indicated that TiCrN, TiAlN, TiAlSiN, and TiAlSiCN formed individually and as a multi-layer of high-quality crystalline layers with mostly cubic structures. The studies on the multi-layers coating morphology, roughness and hardness gave reasonable results as a roughness of 35 nm and a hardness of 32.2 GPa. The coated sweep duck blade tillage tools were tested on the field along with a soil bin to obtain their wear behavior at different traveling distances. The draft force of all blades showed promising results. As the coated layers were worn off, their draft force increased. In comparison with single-layer coatings, the multi-layer structure demonstrated an increase in the useful lifetime of the blades. These characteristics have made these materials good candidates for hard coatings to increase the lifetime of instruments utilized for harsh environments4. Most recently, single layer composite coatings such as TiAlN, TiAlSiN, and TiAlSiCN have been investigated for their well-improved mechanical properties compared to TiN coatings8. Multi-layer coatings have also gained researchers’ interest due to their advantages to single layer coatings12. Durmaz and Yildiz exhibited high-quality TiAlN coatings which had high wear resistance13. Even more, research on TiAlSiN coatings has proved suitability of this composite for anti-wear coating applications15. A research conducted by Shizhi et al. revealed that by adding Si (10–15%) to TiN will form TiSiN thin films leading to layers with higher hardness than TiN16. Other studies also proved that the presence of Si in TiN has a significant role in achieving very high hardness compound with better wear resistance17. These line-of-ceramic composites have proven to be well applied to high-temperature resistance turbine and aircraft engine blades19, high resistance to wear for dry cutting tools23, and other coating applications for molds and dies24, plowshares25 and tillage tools26. The utilized deposition method plays the key important role in achieving high-quality crystalline layers. TiN-based materials have been deposited by many different techniques over the past years. To name a few, Magnetron sputtering32, CVD34, PECVD35, multi-purpose plasma immersion ion implantation37, Plasma focus38, PVD40 and arc-PVD44.Titanium-based composites have been of interest of researchers due to their suitable low friction coefficient, high thermal stability, and resistance to oxidation and wearet al.45 who studied the hardness of the layers by changing the Si content ratio. This material was also deposited by Li, et al.35. Sui, et al.7 research on TiAlN-TiAlSiN double layers also revealed a high level of hardness of the coated tools for cutting applications. Along with research conducted on TiAlSiN, TiAlSi46, TiAlN50 and TiN-TiAlN44 multi-layers have also been deposited by arc-PVD method, which have shown promising mechanical properties. For many years, researchers investigated simple single layer coatings to increase wear and oxidation resistance of cutting tools. Nowadays, studies on hardness, wear, and corrosion resistance are being conducted on more complex and nanocomposite multi-layers to obtain a better performance. Recent investigations on TiAlSiCN multi-layers53 have shown their potential to be applied upon cutting tools to resist high temperature, corrosion and wear, and eventually to increase the useful lifetime of the tools.The arc-PVD method is able to form high-quality thin films at low temperatures. The PVD method has been employed to grow many multi-refractory metal compositions for wear, thermal and corrosion resistance applications. For instance, TiAlSiN thin films were deposited by Yu, 54. Sweep blades are the most exploited soil preparation tools which are used on the combined tillage implements. Tillage tools should be strong enough to resist impact and wear and to work in hard soil conditions54. The case of single ceramic composite coated layers has been previously investigated to reduce wear55. In our previous research on TiN single layer coating, we were able to obtain resealable results in increasing the lifetime of sweep duck blades1. Some of other hardening techniques applied to tillage tools are alumina ceramic coatings56, hard facing57, plasma restoration and hardening58, carbo-vibro-arc hardening method59, and hot stamping processes60.One of the hardening applications of thin film deposition is in agriculture, where wear resistant thin films are deposited on tillage tools to obtain better performance and longer lifetime. Tillage tools are the core soil engaging implements which confront high abrasive wear. The processes by which tillage tools wear evidently include impact, abrasion and chemical actions1. The sweep duck blades (carbon steel CK45) will be coated with TiCrN, TiAlN, TiAlSiN, TiAlSiCN layers using an arc-PVD system with no post-annealing process, before or after the deposition. The first layer, TiCrN, is used as a base layer for other layers with higher hardness to be able to be deposited on top of the carbon steel CK45. The multi-layer has been designed in a way that the hardness of the layers increases from the bottom to the top. The experiments on the coated blades will be carried out at the laboratory and real field conditions to obtain more detailed results for both researchers and manufacturers. In order to examine the performance of the coating, the blades are sent to loamy condition soil. They are tested at four different traveling distances by a tractor, and subsequently, they are tested in a soil bin laboratory condition to measure their draft force. Then, samples from the blades are taken apart to be studied on. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are employed to study the surface morphology and topology of the blades before and after the field experiment. Energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) are exploited to indicate the elements and the crystallinity of the thin films. Additionally, the surface hardness and its roughness are studied by a Vickers hardness monitor and a profilometer. We employ a penetrologger to measure soil mechanical resistance. Soil moisture content is obtained using an oven along with soil texture and chemical properties which are investigated by EC meter, pH meter, Anion-Cation spectrophotometer, and a hydrometer.In this research, we aim to deposit TiCrN-TiAlN-TiAlSiN-TiAlSiCN multi-layer for the first time, and eventually use them to increase the useful life of sweep duck blade tillage tools. With the procedure of stacking four different layers with similar crystal structures, we aim to introduce a multi-layer with higher wear resistance, hardness and longer lifetime than single layer TiN2N, and FeTi through Ti and N bonding to the Fe atoms of the substrate. The second layer, TiAlN, had three crystal structures; c-TiN with (111), (220), and (311) peaks, h-TiAlN with (105), (106), (202), and (206) peaks, and c-AlN with (200) and (220) peaks , (106), and (326)} are crystal structures that formed in the third layer, TiAlSiN. XRD of the final layer TiAlSiCN indicated three structures , and h-TiAlN (105), and (206). The XRD data was processed by an Xpert High Score Plus software. The corresponding data sheet reference codes are 01–087–0631 for c-TiN, 01-070-2981 for c-TiCrN, 01-089-4185 for Fe, 03-065-7743 for FeTi, 00-006-0656 for Fe2N, 01-080-2286 for h-TiAlN, 00-046-1200 for c-AlN, 01-079-2011 for h-Si3N4, and 01-076-2484 for c-TiCN.XRD patterns of all individual layers and the whole multi-layer are shown in Fig. aks Fig. . Due to see Fig. . They arτ = Kλ/βcosθ)62  (FWHM) of the XRD peaks, and θ represents the angles where the peaks are positioned63). The crystallite sizes of the crystal structures formed in the bottom layer were calculated to be 30 nm for c-TiN and 30 nm for c-TiCrN, in TiAlN layer; 12 nm for c-TiN, 6.5 nm for h-TiAlN, and 22 nm for c-AlN, in the third layer, TiAlSiN; 9.8 nm for c-TiN, 11.1 nm for h-TiAlN, and 22.73 nm for h-Si3N4, and in the fourth layer (TiAlSiCN) crystallite sizes where 6.47 nm for c-TiN, 6.14 nm for c-TiCN, and 16.7 nm for c-AlN. The crystallite sizes were also calculated from the XRD data of the multi-layer that were 5.66 nm for c-TiN, 11.11 nm for c-TiCN, 15.2 nm for c-AlN, 16.65 nm for h-Si3N4, and 12.91 nm for h-TiAlN.All crystallite sizes were calculated from full width of half maximum (FWHM) of the XRD peaks by using the Debye-Scherrer equation , roughness root mean squared (Rq), maximum valley depth (Rv), maximum peak height (Rp) and maximum height of the profile (Rt), which determines the approximate thickness of the layer  peak was detected in all layers but its intensity decreased as c-AlN and h-Si3N4 formed in the other layers. This can be the result of the formation of polycrystalline h-Si3N4 in the third layer , showed in Fig. et al. and Peng’s et al. research67. As well as the mentioned structures, h-TiAlN formed without post-annealing  peaks which were not similar to the peaks observed in Fig. The XRD Fig.  data demyer Fig.  and amoryer Fig.  This is ing Fig. . In the x in TiAlSiN thin films10. It is notable that in recent attempts on the growth of TiN-based layers14 XRD patterns were different with much less intense peaks than the data in Fig. et al. study27.It is notable that in comparison with single layers, the crystallite size for all structures has been reduced in the multi-layer and only c-TiCN and h-TiAlN crystallite sizes have been increased. As the results show, the crystallite sizes have also reduced by the presence of Si and Al atoms in the TiAlN, TiAlSiN, and TiAlSiCN thin films. The same issue was reported in previous study where the reason for crystallite size reduction was discussed to be due to the presence of amorphous SiNet al.6 is notable. Kuptsov et al. in a research on TiAlSiCN layers which were deposited by a DC magnetron sputtering and annealed at high temperatures52, could not obtain uniform stacked layers like the ones grown by using the arc-PVD method in our study  was inserted into the chamber that increased its pressure to 10−3 mbar. The coating targets, 99.9% Ti and 99.9% Cr were biased up to 40 V and the deposition was undergone for 80 min to form TiCrN thin film. Following the TiCrN layer formation of about 2.2 μm, the chamber was cleaned with argon (Ar) gas. The second phase of the deposition began with the same working gas (N2), vacuum pressure, biasing voltage, and deposition time, but in this stage for the formation of the second layer a 99.9% TiAl  target was used. For the deposition of the third layer, TiAlSiN after the cleaning procedure by Ar gas, the samples were coated using 99.9% TiAl  and AlSi  targets with the same conditions mentioned in the first 2 stages. Finally, for the fourth layer, TiAlSiCN, the same targets and conditions as the third stage were utilized but in order to add carbon to TiAlSiN, a mixture of methane (CH4) and N2  was inserted as working gas.A cathodic arc evaporation PVD (JHDI/Quad/01 arc-PVD Sevin Plasma Surface Engineering Co.) was used to deposit TiCrN-TiAlN-TiAlSiN-TiAlSiCN multi-layers on carbon steel CK45 substrates (sweep duck blades). Four sweep duck blades were sandblasted with Al2 and a cone apex angle of 60° was used to measure vertical soil mechanical resistance (soil cone index (CI)). The sweep blades were attached on the tillage tool shanks. An ITMCO 399 tractor was used to pull the tillage tool in the field. The experiment conducted at a soil depth of 15 cm, with a tractor forward speed of 6 km/h at four traveling distances of 2, 4, 6 and 8 km. The first blade was taken off at 2 km traveling distance. Afterward, the second blade was taken off at 4 km, the third blade at 6 km and eventually, the final fourth blade at 8 km.A combined tillage tool equipped with the sweep duck blades was used for the field experiments. Experiments were conducted in an agricultural field located at 51° 6′ longitude and 35° 59´ latitude. Soil samples were taken from the experiment field to measure its soil moisture content and soil chemical properties. These properties were measured by a Biochrom Libra S22 spectrophotometer and a Jenway Flame Spectrometer to indicate the sulfate and sodium of the soil. Soil pH and soil texture were measured by Hana 209 pH meter and a hydrometer. The chemical elements are determined using a burette titration along with a Jenway 4510 conductivity meter to monitor the electrical conductivity (EC) range of the soil. A penetrologger  with cone base area of 1 cm2 were cut off the blades. An Ara Research NANOVAC atomic force microscopy (AFM) and a Phenom-XL scanning electron microscopy (SEM) was utilized for surface morphology investigations. Normal X-ray diffraction (XRD) and both regional and multi-spot (Line Scan) energy-dispersive X-ray spectroscopy (EDX) (attached to the SEM device) were used for element detections and crystallographic studies. A Vickers Hardness tester was utilized to measure the hardness of the coated layers. The surface roughness measurements were carried out by Nano Pajouhan Raga Surface Profilometer 50.10.s and AFM. The blades draft force was measured in the soil bin70, at the depth of 15 cm and soil conditions similar to the field.In order to perform a set of analysis, samples of 1 cm1. The soil physical and chemical properties of the experimental field were as given below. The number of total cations was 13.41 milliequivalent/liter (meq/lit)  and total anions was 15.41 meq/lit . Soil electrical conductivity and soil pH were 1.36 ds/m and 7.62, respectively. Soil physical properties including soil texture, moisture content and soil mechanical resistance known as CI were also measured. The percentage of soil particles was 45% sand, 21% clay and 34% silt. The soil texture of the field was then determined as loam according to soil texture triangle71 The multi-layers of TiCrN, TiAlN, TiAlSiN, and TiAlSiCN with the thickness of 4.2 μm have been deposited on CK45 carbon steel for performance improvement of tillage tools (duck blades). The arc-PVD method was able to apply high-quality layers to the samples with no need for post-annealing at high temperatures (XRD and EDX data supported this statement). The sweep blades were tested at both the lab (soil bin) and on the field to investigate their resistance to wear and draft force. With the comparison of the SEM cross-sectional image and the profilometer roughness the worn layers were calculated for most of the samples to be only about ~100 nm. The soil bin draft force experiment showed an increase of draft force as the coated layers wore off after 2 km distance. All in all, tillage tool blades resistance to wear, use full lifetime where increased due to the use of TiCrN-TiAlN-TiAlSiN-TiAlSiCN multi-layer coatings. This operation improvements are owned to the application of arc-PVD technique in deposition of hard multi-layer.


Bifidobacterium are associated with reduced levels of AMR in early life and suggests that probiotic interventions to increase infant Bifidobacterium levels have the potential to reduce AMR in infants. However, this effect is not sustained at year 2 of age in Bangladeshi infants, underscoring the need for more detailed studies of the biogeography and timing of infant AMR acquisition.Infants are vulnerable to an array of infectious diseases, and as the gut microbiome may serve as a reservoir of AMR for pathogens, reducing the levels of AMR in infants is important to infant health. This study demonstrates that high levels of  Bifidobacterium species are important commensals capable of dominating the infant gut microbiome, in part by producing acids that suppress growth of other taxa. Bifidobacterium species are less prone to possessing antimicrobial resistance (AMR) genes (ARGs) than other taxa that may colonize infants. Given that AMR is a growing public health crisis and ARGs are present in the gut microbiome of humans from early life, this study examines the correlation between a Bifidobacterium-dominated infant gut microbiome and AMR levels, measured by a culture-independent metagenomic approach both in early life and as infants become toddlers. In general, Bifidobacterium dominance is associated with a significant reduction in AMR in a Bangladeshi cohort, both in the number of acquired AMR genes present and in the abundance of AMR genes. However, by year 2, Bangladeshi infants had no significant differences in AMR related to their early-life Bifidobacterium levels. A generalized linear model including all infants in a previously published Swedish cohort found a significant negative association between log-transformed total AMR and Bifidobacterium levels, thus confirming the relationship between Bifidobacterium levels and AMR. In both cohorts, there was no change between early-life and later-life AMR abundance in high-Bifidobacterium infants but a significant reduction in AMR abundance in low-Bifidobacterium infants. These results support the hypothesis that early Bifidobacterium dominance of the infant gut microbiome may help reduce colonization by taxa containing ARGs.IMPORTANCE Infants are vulnerable to an array of infectious diseases, and as the gut microbiome may serve as a reservoir of AMR for pathogens, reducing the levels of AMR in infants is important to infant health. This study demonstrates that high levels of Bifidobacterium are associated with reduced levels of AMR in early life and suggests that probiotic interventions to increase infant Bifidobacterium levels have the potential to reduce AMR in infants. However, this effect is not sustained at year 2 of age in Bangladeshi infants, underscoring the need for more detailed studies of the biogeography and timing of infant AMR acquisition. Bifidobacterium species are key members of the infant gut microbiome, and some species are capable of completely dominating the breastfed infant gut (Bifidobacterium is linked to consumption of human milk oligosaccharide (HMO); some species of Bifidobacterium, notably Bifidobacterium longum subsp. infantis, consume HMO more efficiently than others  acquisition because of the relatively low occurrence of AMR in Bifidobacterium  with the notable exception of tet(W)  , 20. Fureriaceae . Therefo animals . The inc animals . The car animals –26. Thisanimals –.Bifidobacterium in early infancy correlated with reduced levels of AMR in the infant gut microbiome and explored if such correlations extended into the weaning period. To do this, this study examined the association of Bifidobacterium levels in early life with the acquisition of AMR both when an infant may be Bifidobacterium dominated and later in life using a cohort from a developing country (Bangladesh) with a high prevalence of Bifidobacterium dominance of the infant gut microbiome  with sufficient DNA for sequencing were included. High-Bifidobacterium samples  were chosen by selecting the highest-Bifidobacterium sample with sufficient DNA remaining for sequencing which would maintain approximate gender balance with the low-Bifidobacterium samples  had samples that consistently fell in the intermediate-Bifidobacterium group, and 1% (3 infants) had samples that were classified as low Bifidobacterium at all time points. In addition, 22% of infants (63 infants) transitioned from a lower level of Bifidobacterium to a higher level: 17% (50 infants) transitioned from the intermediate group at the earliest time point to the high group, 2% (5 infants) transitioned from the low-Bifidobacterium group to the intermediate group, and 3% (8 infants) transitioned from the low group at the earliest time point to the high group at the last time point. Seventeen percent of infants (48 infants) showed decreased Bifidobacterium over time: 15% (42 infants) transitioned from the high group to the intermediate group, 1% (2 infants) transitioned from the high group to the low group, and 1% (4 infants) transitioned from the intermediate group to the low group. Sixteen percent of infants (45 infants) fluctuated between two categories: 13% (37 infants) fluctuated between the high and intermediate groups, 2% (5 infants) fluctuated between the low and intermediate groups, and 1% (3 infants) fluctuated between the low and high groups. The remaining 3% of infants had 1 sample in each of the three categories. Volatility is a normal feature of the infant gut microbiome . As a result, all samples that met the criteria for low Bifidobacterium and had sufficient DNA for WGS were included . These low-Bifidobacterium samples included one sample from each of the 3 infants who exhibited stable low-Bifidobacterium relative abundance, 4 samples from infants who initially had low Bifidobacterium levels and later had high Bifidobacterium levels, 2 samples from infants who transitioned from the low-Bifidobacterium group to the intermediate group, 3 samples from infants who fluctuated between the low and intermediate groups, and a single sample from an infant who had samples in each of the three categories. For high Bifidobacterium, 18 early-life high-Bifidobacterium (>65% relative abundance of Bifidobacterium) samples were sequenced. The majority of the included high-Bifidobacterium samples (13 samples) were from infants with a stable high-colonization pattern, with 3 samples coming from infants who fluctuated between the high-Bifidobacterium and intermediate categories and 2 samples coming from infants who transitioned from the high group to the intermediate group at later time points. This resulted in a total of 31 early-life infant samples for inclusion. Of these 31 infants, 15 infants had a year 2 sample with sufficient DNA available for WGS. High- and low-Bifidobacterium infants were similar in gender and week of early-life sample collection  and the Chao1 richness index . In the later-life samples, there was no significant difference in alpha diversity between infants who were high or low Bifidobacterium in early life by either the Shannon index  or the Chao1 richness index . However, the later-life samples did have a significantly greater alpha diversity by both the Shannon index  and the Chao1 richness index  , with 15 of the 18 high-Bifidobacterium infants delivered by Caesarean section compared to 4 of 13 low-Bifidobacterium infants delivered by Caesarean section.Infant samples were selected for whole-metagenomic sequencing (WGS) from a prior study of vitamin A supplementation and vaccine efficacy based on the level of rly life . As partrly life . Of the  samples  and B. The study  includedcrobiome , and as llection . METAXA2 samples . As the 10.1128/mSphere.00441-18.1FIG S1P value of 0.025 was used for significance. Download FIG S1, PDF file, 0.3 MB.Alpha diversity measured by the Shannon index and the Chao1 richness index calculated using METAXA2 results. (A) Early-life Shannon index. (B) Early-life Chao1 richness index. (C) Later-life Shannon index. (D) Later-life Chao1 richness index. (E) Early versus late Shannon index. (F) Early versus late Chao1 richness index. As two different metrics were used for alpha diversity, a Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium samples from early life. Run 3 contained all year 2 samples. There were no significant differences in merged read depth by run  (P = 0.87); however, there were significantly more contigs generated for low-Bifidobacterium early-life samples than for high-Bifidobacterium early-life samples. Run 3 did have significantly more contigs than runs 1 and 2 , likely related to the increased diversity of the microbiome between the early-life and later-life samples. Bifidobacterium early-life samples than in low-Bifidobacterium early-life samples  . This is consistent with the alpha diversity analysis which found lower diversity in the high-Bifidobacterium samples, as a less diverse microbiome will have fewer different genomes present and will therefore have a shorter total assembly length. If bias exists, the lower number of reads in the low-Bifidobacterium samples will tend to bias results toward finding an increased number of ARGs in high-Bifidobacterium samples. Comparison to the ResFinder database with default settings revealed that high-Bifidobacterium samples in early life had significantly fewer classes of ARGs present than low-Bifidobacterium samples (P = 0.0021) (Bifidobacterium samples had a median of 4 ARG classes present per sample (range of 2 to 8 ARG classes per sample) compared to low-Bifidobacterium samples, which had a median of 7 ARG classes present per sample (range of 3 to 11 ARG classes per sample).The Bangladeshi samples were sequenced in three separate lanes in three separate runs. Runs 1 and 2 contained a mixture of high- and low- = 0.92) . There wsamples) . However 0.0021) . High-Bi10.1128/mSphere.00441-18.2FIG S2Bifidobacterium samples in early life had a significantly higher merged read depth than low-Bifidobacterium samples in early life. This will tend to bias the results toward detecting more ARGs in high-Bifidobacterium samples in the early-life analysis. A P value of 0.05 was used for significance. Download FIG S2, PDF file, 0.1 MB.High-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00441-18.6TABLE S1Table S1, PDF file, 0.1 MB.Bangladeshi sequencing results. Download Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium samples, but only 9 of the classes were present in high-Bifidobacterium samples. The classes present in the low-Bifidobacterium early-life samples but absent in high-Bifidobacterium early-life samples were glycopeptide resistance (present in 2 low-Bifidobacterium samples) and nitroimidazole (present in 1 low-Bifidobacterium sample). All other classes were detected in at least 9 of the 31 samples. Only a single class, MLS, was present in all samples ; phenicol; sulfonamide; tetracycline; trimethoprim; glycopeptide; and nitroimidazole. All 11 of these classes were present in the low- samples .10.1128/mSphere.00441-18.7TABLE S2Bifidobacterium samples. Download Table S2, PDF file, 0.1 MB.Presence of transferable AMR classes in high- and low-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium samples in early life also had significantly fewer transferable ARGs present (P < 0.0001) (P < 0.0001) , and low-Bifidobacterium samples had a median of 20 ARGs per sample . There were a total of 108 ARGs detected by ResFinder in early-life samples. Of these, 103 were present in low-Bifidobacterium samples and 50 were present in high-Bifidobacterium samples , aadA1 (aminoglycoside resistance), cat(pc221) (phenicol resistance), blaLEN12 (beta-lactam resistance), and strA (aminoglycoside resistance). These genes were found in 4 separate samples, with aadA1 and strA detected in the same sample. The five genes found only in high-Bifidobacterium samples stand in contrast to 58 ARGs found only in low-Bifidobacterium samples. Of these 58 genes, 41 were found in only a single low-Bifidobacterium sample, and 11 were found in two low-Bifidobacterium samples. Due to our small cohort size, an ARG present in only one low-Bifidobacterium sample would have a prevalence of 8% in low-Bifidobacterium infants and 3% in all infants. There was a median of 2 unique ARGs per low-Bifidobacterium sample . Two genes were found in three low-Bifidobacterium samples: one was a beta-lactam resistance gene, blaSHV-1, and the other was a tetracycline resistance gene, tet(31). Two genes were found only in 4 of the 15 low-Bifidobacterium samples: one was a beta-lactam resistance gene, blaACI-1, and one was a tetracycline resistance gene, tet(Q). Two genes were found only in 5 of the 15 low-Bifidobacterium samples, an MLS resistance gene, erm(T), and a fluoroquinolone resistance gene, oqxB. There was not a significant difference between ARG count and delivery mode in early life in either the high-Bifidobacterium infants  or the low-Bifidobacterium infants .High- 0.0001) . This di 0.0001) . High-Bi samples . Of the 10.1128/mSphere.00441-18.3FIG S3Bifidobacterium samples. High-Bifidobacterium samples still had significantly fewer ARGs than low-Bifidobacterium samples (P < 0.0001). A P value of 0.025 was used for significance. Download FIG S3, PDF file, 0.1 MB.Differences in number of ARGs per sample divided by read depth per sample in high- and low-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://github.com/akre96/ResistBlast), was used to attempt to predict the taxa of origin of contigs identified as containing an ARG by ResFinder. The family Enterobacteriaceae was the most common predicted origin of ARGs in both high- and low-Bifidobacterium samples, accounting for an average of 49% of the ARGs per sample detected in the high-Bifidobacterium samples and 43% of the ARGs per sample detected in the low-Bifidobacterium samples. In the high-Bifidobacterium samples, other detected families of origin in decreasing average percentage of ARGs per sample were as follows: Enterococcaceae (34% of ARGs on average), Staphylococcaceae (30% of ARGs on average), Streptococcaceae (29% of ARGs on average), Bifidobacteriaceae (19% of ARGs on average), mixed , unknown , and Actinomycetaceae (7.7% of ARGs on average). All families detected in the high-Bifidobacterium samples were also detected in the low-Bifidobacterium samples, but the low-Bifidobacterium samples identified ARGs originating in additional taxa. In the low-Bifidobacterium samples, detected families of origin, in decreasing average percentage of ARGs per sample, were as follows: mixed (20% of ARGs on average), Campylobacteraceae (19% of ARGs on average), Staphylococcaceae (12% of ARGs on average), Enterococcaceae (11% of ARGs on average), unknown (11% of ARGs on average), Moraxellaceae (10% of ARGs on average), Streptococcaceae (10% of ARGs on average), Bifidobacteriaceae (6.0% of ARGs on average), Peptostreptococcaceae (5.9% of ARGs on average), Bacteroidaceae (5.8% of ARGs on average), Flavobacteriaceae (5.0% of ARGs on average), Prevotellaceae (2.8% of ARGs on average), and Actinomycetaceae (2.7% of ARGs on average).NCBI BLAST , in combBifidobacterium in early life and those who were low Bifidobacterium in early life . By year 2, all infants included had <65% relative abundance of Bifidobacterium  in some infants may reflect the fact that by 2 years of age, 80% of Bangladeshi women are still providing some breast milk to their child   was present in 3 samples. Of the 16 ARGs found only in samples from infants who had low Bifidobacterium in early life, 12 were found in only one sample and 4 were found in two samples.There were no significant differences in merged read depth of the later-life (year 2) samples between infants who were high acterium , an expey weaned . The relir child . At yearor ARGs)  and D. Tacterium . Of the Enterobacteriaceae remained the most common predicted origin of ARGs for infants who had high levels of Bifidobacterium in early life, accounting for an average of 38% of ARGs per sample detected in the year 2 samples. For infants who were low Bifidobacterium in early life, a mixed origin (meaning that the top hits from BLAST were from more than one family) was the most common predicted origin of ARGS, accounting for an average of 36% of ARGs per sample at year 2. In the year 2 samples from infants who were initially high Bifidobacterium, the detected families of origin in decreasing order of average per-sample origin were as follows: mixed, unknown , Streptococcaceae, Enterococcaceae, Pasteurellaceae, Prevotellaceae, Lactobacillaceae, Bifidobacteriaceae, Bacteroidaceae, Brachyspiraceae, and Campylobacteraceae. In the year 2 samples from infants who were low Bifidobacterium in early life, the detected families of origin in decreasing order of average per-sample origin were as follows: Enterobacteriaceae, unknown, Enterococcaceae, Streptococcaceae, Peptostreptococcaceae, Prevotellaceae, Bacteroidaceae, Bifidobacteriaceae, Pasteurellaceae, Clostridiaceae, and Pseudomonadaceae.At year 2, Bifidobacterium samples, high-Bifidobacterium samples had a significantly lower abundance of ARGs than low-Bifidobacterium samples  (Bifidobacterium infants (P = 0.31) or the low-Bifidobacterium infants (P = 0.88).The merged reads were then classified as ARGs using the AMR++ pipeline and normalized by the number of reads mapping to 16S rRNA genes to examine both transferable and nontransferable ARGs. = 0.037) . There wBifidobacterium samples were compared for differences at each of these levels as well as for differences in individual ARGs. There were a total of 18 distinct AMR classes detected in the early-life samples using AMR++, leading to a Bonferroni-corrected P value of 0.00278 to detect a significant difference in normalized ARG abundance in class between high- and low-Bifidobacterium samples using a Kruskal-Wallis test. Of these 18 classes, 10 were at higher normalized abundance levels in low-Bifidobacterium early-life samples: aminocoumarins (P = 0.000141), aminoglycosides (P = 0.000987), bacitracin (P = 0.000242), beta-lactams (P = 0.0000127), cationic antimicrobial peptides (P = 0.00186), elfamycins (P = 0.00000582), fluoroquinolones (P = 0.0000588), multidrug resistance (MDR) (P = 0.000122), rifampin (P = 0.0000151), and sulfonamides (P = 0.000181). In addition, the high-Bifidobacterium samples were significantly higher in a single class of AMR in early-life samples, MLS (P = 0.00000503). Because beta-lactam antibiotics are clinically important, to further explore the resistance to beta-lactam present in these samples, the 240 ARGs belonging to the class of beta-lactamases were classified as either transferable (229 ARGs) or nontransferable (11 ARGs) based on whether or not the gene was included in the ResFinder database, and the abundances of both transferable and nontransferable beta-lactamases were compared between high- and low-Bifidobacterium samples using a Kruskal-Wallis test. Both transferable and nontransferable beta-lactam ARGs were significantly increased in low-Bifidobacterium infants. For transferable beta-lactam ARGs, the median normalized abundance was 0.0941 in the low-Bifidobacterium early-life samples but only 0.00171 in the high-Bifidobacterium samples (P < 0.0001). For nontransferable beta-lactam ARGs, the median normalized abundance was 0.0505 for the low-Bifidobacterium samples but only 0.00286 for the high-Bifidobacterium early-life samples (P < 0.0001).Because the MEGARes database organizes ARGs into the acyclical categories of group, mechanism, and class , high- aP value of 0.00104. A total of 13 of these mechanisms were significantly elevated in the low-Bifidobacterium early-life samples: aminocoumarin-resistant DNA topoisomerases (P = 0.000141), aminoglycoside efflux pumps (P = 0.000362), aminoglycoside efflux regulator (P = 0.000348), class A beta-lactamases (P = 0.0000229), EF-Tu inhibition (P = 0.00000582), fluoroquinolone-resistant DNA topoisomerases (P = 0.0000496), multidrug-resistant (MDR) mutant porin proteins (P = 0.0000320), MDR regulator (P = 0.0000874), multidrug efflux pumps (P = 0.000181), penicillin binding protein (P = 0.0000735), rifampin-resistant beta-subunit of RNA polymerase RpoB (P = 0.0000151), sulfonamide-resistant dihydropteroate synthases (P = 0.000181), and undecaprenyl pyrophosphate phosphatase (P = 0.000242). In high-Bifidobacterium early-life samples, only a single mechanism was significantly increased, 23S rRNA methyltransferases (P = 0.00000281); this mechanism belongs to class MLS. There were 178 different AMR groups  detected in the early-life samples, resulting in a Bonferroni-corrected P value of 0.000281 for significance. Of these, 25 groups were significantly higher in the low-Bifidobacterium early-life samples: ACRB (P = 0.0000464), ACRD (P = 0.000248), AMPH (P = 0.0000796), ASMA (P = 0.000106), BACA (P = 0.000242), BAER (P = 0.000211), CPXA (P = 0.0000465), CPXAR (P =0.000110), CRP (P = 0.0000417), EMRB (P = 0.000110), ERMD (P = 0.000276), EMRK (P = 0.000170), EMRR (P = 0.000242), EVGA (P = 0.000182), FOLP (P = 0.000265), GYRA (P = 0.000164), GYRB (P = 0.0000602), HNS (P = 0.0000100), MARR (P = 0.000215), MDTB (P = 0.000248), OMPF (P = 0.0000320), PARC (P = 0.000226), PARE (P = 0.000101), RPOB (P = 0.0000151), and TUFAB (P = 0.00000582). There was also a single group of AMR elevated in the high-Bifidobacterium samples, ERMX (P = 0.00000279), belonging to mechanism 23S rRNA methyltransferases  , ARO3003369 , ARO3004042 , ARO3000518 , ARO3003890 , ARO3000832 , ARO3003288 , ARO3003807 , ARO3003950 , ARO3002818 , ARO3003511 , ARO3000826 , ARO3003317 , and ARO3000263 . There was a single gene that was significantly enriched in the high-Bifidobacterium samples using a Kruskal-Wallis test, ARO3000596 . More genes were identified as significantly different by ShortBRED than by AMR++, likely resulting from the smaller size of CARD, meaning that a less stringent Bonferroni correction was applied to the ShortBRED analysis. Both ShortBRED and AMR++ identified increased ErmX resistance in the high-Bifidobacterium samples. Unlike AMR++, ShortBRED did not identify any beta-lactamases that were significantly enriched in the low-Bifidobacterium samples; however, the specific gene identified as enriched by AMR++ was not included in CARD. ShortBRED identified significant differences in a gene providing resistance to fosfomycin and a gene providing resistance to nitroimidazole, where AMR++ did not identify any differences in resistance to either of these resistance classes; this may relate to the more stringent P value correction used in the AMR++ analysis.To confirm that the AMR++ results were not an artifact of the method, early-life samples were also analyzed using ShortBRED with the Comprehensive Antibiotic Resistance Database (CARD) 2017 pregenerated ShortBRED database , 38. Bec 0.0014) . ShortBR10.1128/mSphere.00441-18.4FIG S4Bifidobacterium samples had significantly fewer ARGs than low-Bifidobacterium samples . A P value of 0.05 was used for significance on this test. Download FIG S4, PDF file, 0.1 MB.Total AMR count in early-life samples as measured by ShortBRED. High-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.725 and P = 0.685, respectively; Kruskal-Wallis test). For the AMR++ pipeline at year 2, only ARGs, groups, mechanisms, and classes that were significantly different in early life were compared. There were no significant differences at year 2 in total normalized ARGs between infants who had high Bifidobacterium and infants who had low Bifidobacterium in early life . Only the ARGs, groups, mechanisms, and classes that were significantly different in early life were tested in later life to minimize multiple-comparison issues. Despite this, there were no remaining significant differences in any specific ARG, group, mechanism, or class at year 2.In the year 2 samples, there was no significant difference in the number of AMR classes or ARGs detected by ResFinder . However, there was a significant increase in the number of both AMR classes and ARGs in the high-Bifidobacterium samples . The AMR++ pipeline had contrasting results: there was no significant difference in AMR abundance between early-life and year 2 samples for high-Bifidobacterium infants . In contrast, there was a significant drop in AMR abundance between early-life and year 2 infants with low levels of Bifidobacterium in early life   .Bifidobacterium were different between the high- and low-Bifidobacterium samples, quantitative PCR (qPCR) was conducted to measure total bacteria, total Bifidobacterium, and total Enterobacteriaceae in all samples where sufficient DNA remained after sequencing. The Enterobacteriaceae family was chosen for qPCR because it was the most common predicted origin of AMR in the ResFinder analysis. This analysis included 28 of the 31 early-life samples and 14 of the 15 year 2 samples. In early life, the high-Bifidobacterium samples had significantly lower total bacteria than the low-Bifidobacterium samples  (P = 0.338) , but there was no significant difference in total bacteria between early life and year 2 in the early-life low-Bifidobacterium infants . However, the total Bifidobacterium qPCR confirmed that in early life the high-Bifidobacterium samples had significantly more Bifidobacterium than the low-Bifidobacterium samples  . While not reaching significance, there was a trend toward a reduction in Bifidobacterium levels in the high-Bifidobacterium group when comparing early-life and year 2 samples . There was also a trend toward increased Bifidobacterium levels in infants in the low-Bifidobacterium group between early life and year 2 . The Enterobacteriaceae qPCR found that high-Bifidobacterium infants in early life had significantly lower Enterobacteriaceae than low-Bifidobacterium infants  . There was no significant difference in Enterobacteriaceae between the early-life and year 2 samples in the infants with low Bifidobacterium in early life , but there was a significant increase in Enterobacteriaceae in the high-Bifidobacterium infants between early life and year 2 . The lower total bacteria and Enterobacteriaceae in the samples with higher levels of Bifidobacterium are consistent with our hypothesis that acid production by Bifidobacterium suppresses the growth of other taxa, potentially allowing niches that might otherwise be filled by AMR-carrying bacteria to remain open during infancy. The qPCR results for total Bifidobacterium levels were significantly correlated with the Bifidobacterium relative abundance calculated from the 16S rRNA gene sequencing data  and with the Bifidobacterium relative abundance calculated from the WGS data . The correlation becomes stronger after dividing the Bifidobacterium qPCR results by the total bacterial qPCR results . The value of the relative abundance calculated from the qPCR data is potentially problematic, as 10 of the 18 high-Bifidobacterium samples were predicted to have a Bifidobacterium relative abundance greater than 100% and 3 samples were predicted to have a Bifidobacterium relative abundance greater than 200%. This may be related to the use of different 16S regions for the two sets of primers. In addition, higher total Bifidobacterium levels in early life correlated with reduced total normalized AMR abundance , but there was no significant correlation between either Enterobacteriaceae and total normalized AMR abundance  or total bacteria and total normalized AMR abundance .To further confirm that levels of 0.00402) , but in = 0.338) . The inf 0.0001) . There w0.00961) . By year10.1128/mSphere.00441-18.5FIG S5Bifidobacterium samples have significantly more total bacteria than high-Bifidobacterium samples in early life . There was no significant difference in total bacteria between the early-life high-Bifidobacterium and the early-life low-Bifidobacterium infants at year 2 . The early-life high-Bifidobacterium infants experienced a significant increase in total bacteria from early life to year 2 . There was no significant difference in total bacteria between the early and year 2 samples for early-life low-Bifidobacterium infants . (B) Log-transformed concentration of Bifidobacterium (CFU per milliliter). Early-life high-Bifidobacterium samples have significantly more Bifidobacterium than early-life low-Bifidobacterium samples . However, there was no significant difference in Bifidobacterium levels at year 2 between infants who were high Bifidobacterium in early life and those who were low Bifidobacterium in early life . Comparing high-Bifidobacterium samples with both an early-life and a year 2 sample, there was a trend toward lower levels of Bifidobacterium at year 2 . Comparing low Bifidobacterium samples with both an early-life and a year 2 sample, there was a trend toward higher levels of Bifidobacterium in the year 2 samples . (C) Log-transformed concentration of Enterobacteriaceae (CFU per milliliter). Early-life high-Bifidobacterium samples have significantly less Enterobacteriaceae than early-life low-Bifidobacterium samples . However, there was no significant difference in Enterobacteriaceae levels at year 2 between infants who were high Bifidobacterium in early life and those who were low Bifidobacterium in early life . There was no significant change in Enterobacteriaceae levels between early life and year 2 in low Bifidobacterium infants , but there was a significant increase in Enterobacteriaceae levels between early life and year 2 in high Bifidobacterium infants . A P value of 0.0167 was used for significance. Download FIG S5, PDF file, 0.2 MB.qPCR results. (A) Log-transformed concentration of total bacteria (CFU per milliliter). Low-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium correlate with a lower trimmed read depth , which will bias the results away from the null hypothesis. There was no significant correlation between Bifidobacterium and read depth at month 12 . The AMR++ pipeline analysis does include a normalization step that adjusts for read depth. Bifidobacterium levels and total AMR including all infants at month 4, total AMR was log transformed. A regression model was then run with the log-transformed total AMR as the outcome variable and the Bifidobacterium level in the sample, the number of times that an infant received antibiotics before age 4 months, and delivery mode as predictor variables using the glm command in R 3.4.3. Relative abundance of Bifidobacterium in the sample was significantly associated with the log-transformed total AMR , while the number of times that an infant received antibiotics (P = 0.657) and delivery mode (P = 0.24) were nonsignificant. The association between Bifidobacterium and the log of the total AMR remained significant after removing antibiotic exposure and delivery mode from the model . This is consistent with increasing levels of Bifidobacterium correlating with decreasing levels of AMR. However, as only 5 infants of the 100 Swedish infants ever received antibiotics, power may be limited to detect the actual influence of antibiotic exposure on AMR level.To confirm the findings of AMR reduction detected in the Bangladeshi cohort, the month 4 and month 12 samples from the study by Bäckhed et al.  of infan10.1128/mSphere.00441-18.9TABLE S4Table S4, PDF file, 0.2 MB.Swedish cohort sequencing results. Because of the larger insert size, reads were not merged. Download Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Bifidobacterium while 48 met the criteria for low Bifidobacterium  , with low-Bifidobacterium infants having significantly higher levels of tetracycline resistance.Of the 100 infants at month 4, only 6 met the criteria for high acterium . At mont 0.0613) . In thisBifidobacterium, resulting in a Bonferroni-corrected P value of 0.00106 for comparisons. Only two mechanisms were significantly enriched in month 4 low-Bifidobacterium samples: class A beta-lactamases (P = 0.000347) and tetracycline resistance ribosomal protection proteins (P = 0.000153). No AMR mechanisms were significantly enriched in high-Bifidobacterium samples. There were 173 different AMR groups present in the month 4 high- or low-Bifidobacterium samples, resulting in a Bonferroni-corrected P value of 0.000289 for significance. There was one group significantly increased in the low-Bifidobacterium samples, TETQ (P = 0.0000467). There were 599 different ARGs detected in the month 4 high- and low-Bifidobacterium samples, resulting in a Bonferroni-corrected P value of 0.0000835. No individual ARGs differed significantly between the high- and low-Bifidobacterium month 4 samples.There were a total of 47 AMR mechanisms detected in the month 4 samples classified as high or low Bifidobacterium for all infants had fallen below 30%, and below 20% for all infants except one  , despite an increase in tetracycline abundance in the high-Bifidobacterium samples between months 4 and 12  and a decrease in tetracycline abundance in the low-Bifidobacterium samples between months 4 and 12  ; however, month 4 low-Bifidobacterium infants had a significantly higher abundance of the mechanism tetracycline resistance ribosomal protection proteins than month 4 high-Bifidobacterium infants (P < 0.0001).By month 12, the relative abundance of cept one . The one 0.0105) . Because 0.0465) . Only thBifidobacterium infants did not have significantly different abundances of total AMR . However, as with the Bangladeshi cohort, low-Bifidobacterium early-life infants did experience a significant drop in the abundance of AMR between month 4 and month 12   .Bifidobacterium at levels greater than 65% correlates strongly with reduced levels of ARGs, in terms of both a reduced diversity of transferable ARGs present and reduced ARG abundance in the Bangladeshi infants. The relative abundance differences in Bifidobacterium do correlate with differences in absolute abundance of Bifidobacterium by qPCR. In addition, the high-Bifidobacterium infants had lower levels of total bacteria and of family Enterobacteriaceae than the low-Bifidobacterium infants. Despite being the most common predicted origin of AMR, levels of family Enterobacteriaceae measured by qPCR did not correlate with a reduction in total AMR abundance in early life. This may be related to the small sample size of this analysis (only 28 samples had sufficient DNA for qPCR). That there was a significant correlation between Bifidobacterium and a reduction in total AMR abundance suggests that Bifidobacterium may suppress more AMR-carrying taxa than just Enterobacteriaceae. This is consistent with past research that finds that Bifidobacterium has an inhibitory effect on the growth of a variety of other commensals, including pathogens, via organic acid (particularly acetate) and bacteriocin production (–Bifidobacterium infants (P = 0.06). A key difference is that in the Swedish cohort the typical relative abundance of Bifidobacterium in early life was much lower, resulting in very few infants in the high category for the comparisons. However, a regression model testing for the association between total ARG abundance and Bifidobacterium level using all infants in the Swedish cohort did find a significant association, where higher levels of Bifidobacterium correlated with lower levels of total ARG abundance. This suggests that infants with gut microbiomes dominated by Bifidobacterium are at lower risk of AMR-associated diseases, although additional studies are needed to confirm this hypothesis.AMR is a major public health challenge, and the gut microbiome may come to harbor ARGs even in the first few days of life , 29. Glooduction –43. ThisBifidobacterium  correlates with improved vaccine responses at 2 years .Historically, the infant microbiome was dominated by acterium , 45. Hownt stool . Others ocations , suggestulations , 47 and ulations , 49, amoBifidobacterium levels are associated with reduced beta-lactam resistance in early-life samples in Bangladesh. Beta-lactams such as amoxicillin are the first-line antimicrobials for acute otitis media  or from year 2 (Bangladeshi infants) were available, and the earlier timing of the Swedish samples may explain the discrepancy in whether differences in later-life total ARG abundance could be detected by early-life Bifidobacterium levels. The limited time points of collection in both the Swedish and Bangladeshi cohorts do not provide the data needed to fully describe the trajectory of AMR colonization in these infants or to explore how other environmental factors affect AMR colonization. An additional limitation of this study is a lack of data on antimicrobial use in the Bangladeshi cohort. It is possible that the infants with low Bifidobacterium received antimicrobials, which may drive an increase in AMR in the microbiome. To fully understand the relationship between Bifidobacterium dominance in infancy and AMR levels in early life, studies with dense sampling during infancy and childhood including infants from both developed and developing countries are needed to first understand how stability or fluctuations of Bifidobacterium dominance affect AMR levels and then to understand how far into the weaning process and under what environmental conditions a reduction in AMR associated with early high levels of Bifidobacterium may extend.While high bifidobacterial levels correlated with reduced AMR during early life, there were mixed results in the effect of Bifidobacterium levels in early infancy and vaccine response in early infancy and at age 2 years (NTC02027610) (Bifidobacterium (<20% relative abundance) or high in Bifidobacterium (>65% relative abundance) in early life  for whole metagenomics sequencing. Only a single early-life sample per infant was included. The high prevalence of Bifidobacterium dominance of the gut microbiota in this cohort resulted in a limited number of low-Bifidobacterium samples available for inclusion. In addition, reliable information on whether infants received antimicrobial agents was unavailable and so could not be included in analysis.This study used samples collected from infants enrolled in a clinical trial of vitamin A supplementation and vaccine effectiveness  and a follow-up study on 2027610) , 52. TheDNA was extracted as described in the work of Huda et al.  and sentBifidobacterium samples from early life, while the third run contained the samples from year 2.Samples were sequenced in three separate sequencing runs. The first two runs consisted of a mix of high- and low-Organisms may have intrinsic, adaptive, or acquired AMR . IntrinsBifidobacterium infants in early life were compared in R 3.4.3 statistical software  (https://github.com/akre96/ResistBlast) to identify the most probable taxon of origin for each ARG. For each ARG, the contig containing the ARG was run through NCBI BLAST, and the top three species hits with at least 90% identity match were returned. As ResFinder is focused on transferable ARGs which can be shared across taxa, the family of each of the top three hits was identified. If all three hits were from the same family, the ARG was considered to originate in that family. If the top three hits originated from two or more families, the origin or the ARG was considered mixed. If ResistBlast returned no hits or if all hits were to uncultured bacterium, the family of origin was considered to be unknown. The percentage of ARGs in each sample mapping to each origin was calculated by dividing the number of ARGs mapping to a particular origin in each sample, dividing by the total number of ARGs in that sample, and multiplying by 100.Prior to analyzing reads with ResFinder or MEGARes, host subtraction was completed on each sample using BMTagger in BMTools v. 1  and the software  using Kr (nr/nt)  using a n is each individual ARG in the MEGARes database, N(AMR) is the number of reads mapping to ARGs according to AMR++, L(reads) is the length of the sequencing reads and cancels out of the equation because the length is the same for both the AMR reads and the 16S reads, L(AMRref) is the length of the AMR reference gene in the MEGARes database, N(16S) is the number of reads mapping to 16S rRNA gene bacterial reads by METAXA2, and 1,432 is the average length of a 16S rRNA gene sequence in the Greengenes database. Differences in total AMR abundance between high- and low-Bifidobacterium infants in early life were tested in both early-life samples using the Kruskal-Wallis test in R  was used to test for an association between AMR levels and delivery mode in early life. To confirm the results of the early-life Bangladeshi comparison, FLASH merged reads were also processed using ShortBRED  and the MEGARes v 1.01 database using default settings  to deterest in R . An advahortBRED  using deSequencing results from year 2 samples were processed in the same manner as early-life samples for the ResFinder analysis. For the abundance analysis, only the classes, mechanisms, groups, and genes significantly different in early-life samples were compared in later-life samples to determine if any differences in AMR in early life are maintained at later time points. Early-life and year 2 total abundances of AMR were then compared using a Wilcoxon paired test.Bifidobacterium. Briefly, DNA extracted from stool was diluted 1:10 before use. The standard curves for qPCR were generated using a 10-fold dilution series of DNA extracted from Escherichia coli sldp 38-1  or Bifidobacterium longum subsp. infantis ATCC 15697  in liquid cultures at late exponential phase for which cell numbers were determined by quantitative culture. Total bacterial qPCR was completed using a melting temperature (mT) of 60°C using the 27F (5′AGAGTTTGATCCTGGCTCAG3′) and 355R (5′CTGCTGCCTCCCGTAGGAGT3′) primers described by Cariveau et al. (Bifidobacterium qPCR was completed using a mT of 58°C using the genus Bifidobacterium F (5′TCGCGTC[C/T]GGTGTGAAAG3′) and genus Bifidobacterium R (5′CCACATCCAGC[A/G]TCCAC3′) primers described by Krumbeck et al. (Enterobacteriaceae qPCR was completed using a mT of 56°C using the family Enterobacteriaceae F (5′CATTGACGTTACCCGCAGAAGAAGC3′) and family Enterobacteriaceae R (5′CTCTACGAGACTCAAGCTTGC3′) primers as described by Oh et al.  and normalized total AMR abundance (from AMR++) was calculated using Spearman’s test. Early-life and year 2 total bacteria and total Bifidobacterium levels were stratified by Bifidobacterium level, and only infants with both an early-life and a year 2 sample were included for statistical testing using a Wilcoxon paired test.Quantitative PCR (qPCR) was conducted to determine the absolute abundance of total bacteria and total u et al. . Total Bk et al. . Total Eh et al. . Total aERP005989. Briefly, this cohort enrolled 100 mother-infant dyads and used WGS to examine the development of the infant microbiome from birth until age 12 months. For this study, the infant month 4 and month 12 samples were included. After downloading the month 4 and 12 sequencing files, data were processed using AMR++, which includes a normalization step to adjust for differences in read depth, with the same criteria as the Bangladeshi cohort except that the FLASH merge step was skipped due to a longer insert size. As samples were sequenced without selection based on Bifidobacterium levels, correlation between read depth and Bifidobacterium relative abundance was tested using Spearman’s correlation test at both months 4 and 12. Then, regression using the log-transformed total AMR was completed in R 3.4.3 using the glm command and including the number of times that infants that were exposed to antibiotics before age 4 months and the relative abundance of Bifidobacterium in the month 4 samples as predictor variables. Then, to more directly compare results of the two cohorts, samples were classified as high or low Bifidobacterium using the METAXA2 results on the month 4 samples using the same criteria as for the 16S rRNA gene sequencing on the Bangladeshi cohort. The abundances of AMR in the month 4 and month 12 samples were calculated using the AMR++ pipeline as described above.To confirm findings in the Bangladeshi cohort in an unrelated cohort, samples from the study by Bäckhed et al.  of the hSRP133760.Host subtracted data are available on the NCBI SRA under accession number 10.1128/mSphere.00441-18.10TABLE S5Bifidobacterium samples in Sweden. P value for significant difference at the class level was 0.00294, at the mechanism level was 0.00106, and at the group level was 0.000289. Download Table S5, PDF file, 0.1 MB.Median normalized abundance for AMRs identified by MEGARes that were significantly different between the high- and low-Copyright © 2018 Taft et al.2018Taft et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Latrodectus tredecimguttatus has poisonous components not only in venomous glands but also in eggs. Our previous work had carried out a transcriptome analysis of the spider eggs in an attempt to probe into the molecular basis of the egg toxicity. A proteinaceous toxin, named Latroeggtoxin-V, was mined from the identified transcriptome. In this study, the gene of Latroeggtoxin-V was cloned and heterologously expressed, and the anticancer activity of the recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was characterized. Activity assay found that rLatroeggtoxin-V could selectively act on breast cancer line MDA-MB-231 cells, not only arresting their cell cycle, inhibiting their proliferation and migration, but also inducing their apoptosis. Bioinformatics analysis suggested that Latroeggtoxin-V belongs to the ATPase inhibitor protein family and the further activity assay showed that the rLatroeggtoxin-V inhibited the activity of the Na+/K+-ATPase in MDA-MB-231 cells in a concentration-dependent manner, suggesting that the anticancer activity of Latroeggtoxin-V is based on its affecting the ion transport and receptor functions of Na+/K+-ATPase. The present work not only laid the foundation for the utilization of Latroeggtoxin-V in the anticancer drug development and the related fields, but also provided a new paradigm for exploration of the proteinaceous toxins under the direction of transcriptomics and bioinformatics.As a black widow spider,  Latrodectus tredecimguttatus, also known as the “black widow”, belongs to Arthropoda, Arachnoidea, Araneida, Theridiidae, Latrodectus in zoology and is one of the most poisonous spiders known in the world [L. tredecimguttatus can release highly toxic venom, causing severe pain in the whole body after being stung by it and leading to functional or organic diseases of single or multiple organs such as liver, brain, kidney, heart and lung, and can even lead to death [L. tredecimguttatus contain many toxic components [+ channel current in rat dorsal root ganglion neurons, showing toxicity toward both mice and Periplaneta americana [Spider he world ,2. L. trto death ,4. Furthmponents ,6,7,8,9,mponents ,10,11,12mponents . By compmponents . Latroegmericana . Latroegmericana . No doubL. tredecimguttatus eggs, from which 280 open reading frames encoding possible proteinaceous toxins were identified and the biological functions of the encoded toxins were bioinformatically predicted [At present, various omics strategies have successively emerged in the field of life science, of which transcriptomics based on the second generation high-throughput sequencing has been widely used in gene expression analysis. With this technique, the researchers can comprehensively and rapidly obtain the genomic transcription information of the researched object, which is helpful for revealing the molecular basis and mechanism underlying different biological characteristics ,18. Our redicted , thus prredicted , suggestBioinformatic analysis on Latroeggtoxin-V showed that the theoretical molecular weight (MW) and isoelectric point (pI) of this protein were 10.17 kDa and 6.21, respectively. The prediction of secondary structure indicated that two kinds of secondary structure units were contained in the Latroeggtoxin-V molecule: α-helix and coil. The secondary structure was dominated by α-helix formed by C-terminal sequence, accounting for 68.1% of the total sequence, while the remaining 21.9% was in the coil conformation A. The se260 nm/OD280 nm = 1.9), with a concentration of 900 μg/mL. According to the results of previous transcriptomic analysis of the eggs of L. tredecimguttatus, the nucleic acid sequence encoding Latroeggtoxin-V as well as amino acid sequence was obtained (Latroeggtoxin-V-F and Latroeggtoxin-V-R) were designed and used for PCR reaction, using the cDNA prepared by reverse transcription from the extracted total RNA as the template. Agarose gel electrophoresis and sequencing analysis showed that the PCR product was a 310-bp gene fragment that was consistent with that predicted , indicating that the inhibitory effect of rLatroeggtoxin-V on the proliferation in MDA-MB-231 breast cancer cells has selectivity. The IC50 of rLatroeggtoxin-V against MDA-MB-231 cells was calculated to be about 43 μM.After the MDA-MB-231 and HEK293 cells were treated with different concentrations of rLatroeggtoxin-V  for 48 h, the cell viability was assessed by MTT assay. The results  showed tIn order to investigate whether rLatroeggtoxin-V, besides inhibiting the proliferation in the MDA-MB-231 cells, can decrease the viability of MDA-MB-231 cells through inducing apoptosis, apoptotic morphology of the cells treated with 40 μM rLatroeggtoxin-V and untreated cells was comparatively observed after Hoechst 33258 staining. The results showed that the karyopyknosis characteristic of apoptosis appeared in some cells treated with rLatroeggtoxin-V for 48 h A, but nop < 0.01)  . This re0/G1 phase increased with the increase of rLatroeggtoxin-V concentration. When the concentrations of rLatroeggtoxin-V were 50 μM and above, the percentage of MDA-MB-231 cells in G0/G1 phase was significantly higher than that of the control (p < 0.05). Correspondingly, the percentage of the MDA-MB-231 cells in S phase was significantly lowed, and that of the MDA-MB-231 cells in G2/M phase was not significantly changed. These results indicate that rLatroeggtoxin-V could induce cell cycle arrest at G0/G1 phase in the MDA-MB-231 cells.As shown in +/K+-ATPase activities were determined using a Na+/K+-ATPase assay kit . The results showed that rLatroeggtoxin-V could in vitro inhibit the activity of Na+/K+-ATPase in a concentration-dependent manner  of control (p < 0.05).After the MDA-MB-231 cells were treated with different concentrations of rLatroeggtoxin-V  for 0 and 24 h, the cells were separately harvested and their Nat manner . When rLL. tredecimguttatus egg transcriptome that encodes a protein: Latroeggtoxin-V that had not been isolated from the eggs due to its too low abundance and the complexity of the egg protein composition. BLAST analysis suggested that Latroeggtoxin-V might have activities of inhibiting Na+/K+-ATPase, anticancer and antibacterium (Staphylococcus aureus and Bacillus subtilis (data not shown), could inhibit the Na+/K+-ATPase, proliferation and migration of breast cancer cell line MDA-MB-231 cells, and induce their apoptosis.The conventional strategy for obtaining and studying the desired proteinaceous toxins and other bioactive components is to directly extract them from natural materials followed by activity screening. This strategy has limitations such as high cost, low yield and long period, particularly when the natural material is limited, the material composition is complex, and the desired component is low-abundant. Next-generation sequencing-based transcriptomics provides an effective means to characterize the gene expression products. Theoretically, it is now possible to have virtually all the mRNAs in a sample to be identified and analyzed, which is suitable for rapidly obtaining the inventory of all proteinaceous components without the need for a reference genome. Transcriptome provides much information on the full-length as well as partial gene sequences encoding proteins and peptides, including those expressed in such a low abundance that it is impossible for them to be directly purified from the natural materials. The contemporary bioinformatics may predict the potential biofunctions of the expressed products, and thus narrow the range of activity screening. In our present study, we cloned and heterologously expressed a unigene selected from the acterium . The exp+/K+-ATPase is a multiple-transmembrane plasma membrane protein complex that utilizes the energy from the hydrolysis of ATP to drive 3 Na+ out of cells and 2 K+ into cells, which is essential for normal resting membrane potential and maintaining the electrolyte and fluid balance in cells, organs and whole body [+/K+-ATPase leads to depletion of intracellular K+ and accumulation of intracellular Na+, which in turn results in membrane depolarization and increase in cytosolic free Ca2+ concentration due to activation of voltage-gated Ca2+ channels and reversed operation of the Na+/Ca2+ exchanger [+/K+-ATPase also works as a cell surface receptor and a key player of cell adhesion, participating in modulation of cell signaling mechanism, affecting proliferation, cell–cell interaction, differentiation and apoptosis, etc. [+/K+-ATPase is a house-keeping enzyme with multiple specialized functions and is required for the normal physiological and biochemical activities even the survival of all cells [+/K+-ATPase are related to the development and progression of breast cancer, the most prevalent malignancies found in women all over the world [+/K+-ATPase, this enzyme could be used as a potentially important target for the development of anti-breast cancer drugs. Furthermore, the Na+/K+-ATPase can serve as a target for a number of ligands, thus providing even more possibilities for screening specific anti-breast cancer drugs [+/K+-ATPase and possess potent anti-breast cancer activity, displaying potential in the development of anti-breast cancer drugs [+/K+-ATPase-mediated Na+/K+ transport [+/K+-ATPase in human breast cancer line MDA-MB-435s, and had no effect on cell viability, but inhibited proliferation by activating the Na+/K+-ATPase-mediated signaling pathways that led to the increase in the level of cell cycle inhibitor and the growth arrest [The Naole body ,24,25. Ixchanger ,27. Calcxchanger . In addiis, etc. ,30,31. Ger drugs ,33,34. Fransport . Kometiah arrest . Howeverh arrest ,38,39,40Apis mellifera, has been shown to suppress EGF-induced cell motility and invasion by inhibiting PI3K/Akt/mTOR signaling pathway in breast cancer cells [Tityus discrepans and found to induce apoptosis of human breast carcinoma SKBR3 cells but has a negligible effect on non-malignant MA104 monkey kidney cells [Dendroaspis angusticeps (Elapidae) shows strong cytotoxicity against breast adenocarcinoma MDA-MB-231 cells [L. tredecimguttatus egg transcriptomic data with the help of bioinformatics. The experimental results showed that the rLatroeggtoxin-V at the lower tested concentrations could induce apoptosis of breast cancer MDA-MB-231 cells  at a concentration of 1 mM . The conserved domain and homologous sequence of Latroeggtoxin-V was searched by the CCD database (https://www.ncbi.nlm.nih.gov/cdd/) and BLASTP program (https: //blast.ncbi.nlm. nih.gov/Blast.cgi) in NCBI. PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/) was used to analyze the secondary structure, and on this basis, the hydrophobic property and coiled-coil of α-helix in Latroeggtoxin-V were analyzed by Heliquest (http://heliquest.ipmc.cnrs.fr/) and COIL Server .Some bioinformatics online software was used to analyze the Latroeggtoxin-V. The molecular weight (MW) and isoelectric point (pI) were analyzed by compute pI/MW tool of ExPASy  was used to extract the total RNA of the spider eggs 1–2 weeks before hatching. The purity, integrity and concentration of the extracted total RNA were detected by agarose gel electrophoresis and biophotometer , respectively. According to the instructions in the manual of SuperscriptTMIII kit , the total RNA was reversely transcripted to cDNA. Based on the Latroeggtoxin-V gene sequence obtained by the transcriptomic analysis of the eggs, a pair of gene-specific amplification primers were designed  and 36.5 μL ddH2O. The PCR products were gel-purified using the QIAquick® Gel Extraction Kit  and ligated into the pMD19-T vector after being added with a poly (A) tail. The ligation products (named pMD19-T-Latroeggtoxin-V) were transformed into DH5α competent cells, and the transformed cells were spread on a LB solid plate containing 100 μg/mL ampicillin. The next day, the colonies growing on the plate were picked for colony PCR detection. The positive colonies were sequenced by sequencing company to identify which contained the correct sequence.Trizol reagent  from correctly sequenced bacteria, and then the plasmids and pET-28a expression vectors were simultaneously digested with restriction endonuclease FastDigest BamHI and FastDigest XhoI . After gel-purification and ligation of the digested products with T4 ligase , the constructed recombinant vectors pET-28a-Latroeggtoxin-V were transformed into BL21 (DE3). The transformed strains were plated on LB solid medium containing 100 μg/mL Kanamycin, and single colonies was selected for colony PCR detection after culturing at 37 °C for 16 h. The plasmids extracted from the colonies with positive colony PCR results were analyzed by double enzyme digestion and sequencing, and the strains with the correct analysis results were inoculated into the LB liquid medium containing 100 μg/mL Kanamycin and shaken at 37 °C and 225 r/min overnight. The next day, the bacterial solution was inoculated into 50 mL of new LB liquid medium at a ratio of 1:50, which was shaken at 37 °C and 225 r/min until the OD600 value reached 0.6–0.8. Prokaryotic expression was induced by the addition of IPTG  to a final concentration of 0.25 mM. After 5 h of inducing incubation, SDS-PAGE was performed with a 12% SDS-polyacrylamide slab gel to detect the induced expression of Latroeggtoxin-V fusion protein.The pMD19-T-2PO4, 300 mM NaCl, 50 mM imidazole, pH8.0). The bacterial suspension was treated with the Ultrasonic crusher JY99-IIDN , and then centrifuged at 10,000 g for 30 min to collect the supernatant. For purifying Latroeggtoxin-V fusion protein, the supernatant was made to pass through a nickel-NTA agarose column  pre-equilibrated with the native lysis buffer. The non-target proteins binding to the nickel-NTA agarose column were washed sequentially using 20 mL native lysis buffer and washing buffer . Latroeggtoxin-V fusion protein with His-tag was eluted with 40 mL elution buffer , followed by desalination and concentration with an ultrafiltration tube Millipore UFC901096 . SDS-PAGE was used to detect the purity of the prepared Latroeggtoxin-V fusion protein sample.After expanding culture and inducing expression, the bacterial culture was centrifuged at 6000 g for 5 min to collect the precipitate, which was then resuspended in a native lysis buffer . The cleaved products were eluted with a linear gradient of acetonitrile containing 0.1% TFA from 0% to 70% in 40 min at a flow rate of 1.0 mL/min. The molecular weight of the components in each elution peak was determined by electrospray ionization mass spectrometry  and thus the peak containing recombinant Latroeggtoxin-V (rLatroeggtoxin-V) was selected and lyophilized.The Latroeggtoxin-V fusion protein was cleaved by enterokinase at 25 °C overnight in a cleavage buffer  at an enzyme: substrate ratio of 1:50 . The cleaved products were separated with RP-HPLC  using a C18 column  containing 10% fetal bovine serum  at 37 °C, 5% CO2 and a relative humidity of 90%, using a carbon dioxide incubator . When the cells were cultured to exponential phase, they were dissociated with trypsin digestion , collected and then seeded in a 96 well plate at a concentration of 103 cells/well. After incubation for 24 h, the medium was removed by aspiration and replaced with 100 μL of experimental medium containing 5 different concentrations of rLatroeggtoxin-V , each of which was plated into 4 wells. After the toxin treatment was performed for 48 h, the cell viability was assessed using the 3--2,5-diphenyltetrazolium bromide (MTT) assay . Ten μL of MTT reagent was added to each well and the plates were incubated in the dark for 4 h at 37 °C. Subsequently, the absorbance of MTT was measured at 490 nm by a Varioska Flash plate reader . The relative cell viability was calculated using the following equation: Atest/Acontrol × 100%, where ‘Acontrol’ is the absorbance of the control (0 μM rLatroeggtoxin-V treatment) and ‘Atest’ is the absorbance of the tests.Human embryonic kidney 293 (HEK293) cells, non-tumor-derived cells used as control, and breast cancer cell line MDA-MB-231 cells  were cultured in DMEM/high glucose medium (Gibco6 cells/well), and divided into two groups: Control groups (0 μM rLatroeggtoxin-V treatment) and test groups of (40 μM rLatroeggtoxin-V treatment). After treatment for 48 h, the cells were fixed with 4% formaldehyde for 10 min and washed in PBS twice. The cells were then stained with 0.5 mL of Hoechst 33258  for 5 min, washed in PBS twice, and photographed with an Olympus IX83 fluorescence microscopy  at 360 nm.Hoechst 33258 staining strategy was used to observe the apoptotic morphology of the cells treated with rLatroeggtoxin-V. MDA-MB-231 cells were seeded in the 6 well plate , each of which was plated into 3 wells. The wound widths at 0 and 24 h after scratching were measured and photographed, respectively, with a DMLB2 microscopy  to determine the effect of rLatroeggtoxin-V on the cell migration.Wound-healing assay was used to assess the effect of rLatroeggtoxin-V on MDA-MB-231 cell migration in culture. A 24-well plate was seeded at 106 cells /mL. After adhering to the wall, the cells were cultured in serum-free medium for 12 h so that they could grow synchronously. Different concentrations of rLatroeggtoxin-V  were used to treat the cells for 48 h, and then the cells were harvested, washed in cold PBS and fixed in 75% alcohol at 4 °C for 12 h. The fixed cells were resuspended in PBS containing 0.25 mg/mL RNase A and 0.05 mg/mL propidium iodide (PI) for 30 min, and analyzed using flow cytometry .The effect of rLatroeggtoxin-V on the cell cycle of MDA-MB-231 cells was assayed according to the method of Sun et al. . Briefly+/K+-ATPase in MDA-MB-231 cells was assayed as described [6 cells/well in 1.8 mL volume and cultured overnight. Next day, the medium was removed by aspiration and replaced with 1.8 mL experimental medium containing 4 different concentrations of rLatroeggtoxin-V , each of which was plated into 3 wells. After treatment for 12 h, the cells were harvested by trypsinization with 0.05% trypsin and suspended in a PBS buffer. The cells were disrupted by ultrasound , and the supernatant containing Na+/K+-ATPase was collected by 8000 g centrifugation at 4 °C for 10 min. The Na+/K+-ATPase activity was evaluated with a Na+-K+-ATPase detection kit  according to the instructions of the manufacturer. The concentration of inorganic orthophosphate (Pi) liberated from the hydrolysis of ATP was spectrophotometrically measured at 660 nm. The amount of Na+/K+-ATPase decomposing ATP to produce 1 micromole Pi per milligram of cell protein per hour is one enzyme activity unit. The protein concentration was determined by the BCA (bicinchoninic acid assay) method  with human serum albumin as a standard.The activity of the Naescribed ,51,52. Mp < 0.05.Data were expressed as mean ± SD. Experimental data were analyzed using SPSS v19.0 statistical software  with the least significant differences between samples being


The comparison of estimated step lengths to reference measurements showed no significant difference (p > 0.05) and acceptable agreement . Step length estimations further improved  after smoothing the original estimated step lengths with a third order polynomial function (R2 = 0.94 ± 0.04). In conclusion, during maximal sprint acceleration, acceptable estimates of stride and step length were obtained from IMU-derived step times and 30-m (split) and 60-m sprint times. During sprint acceleration, step length, step rate, ground contact, and airtime are key variables for coaches to guide the training process and technical development of their athletes. In the field, three of these variables are easily obtained with inertial measurement units (IMUs), but, unfortunately, valid estimates of step length with IMUs currently are limited to low speeds (<50% max). A simple method is proposed here to derive step length during maximal sprint acceleration, using IMUs on both feet and two timing gates only. Mono-exponential velocity-time functions are fitted to the 30-m (split) and 60-m times, which in combination with IMU-derived step durations yield estimates of step length. To validate this approach, sixteen well-trained athletes with IMUs on the insteps of both feet executed two 60-m maximal sprints, starting from a three-point position. As a reference, step lengths were determined from video data. The reference step lengths combined with IMU-derived step durations yielded a time series of step velocity that confirmed the appropriateness of a mono-exponential increase of step velocity (R In many sports, sprint acceleration is essential for good performance. Sprint speed is the product of step rate and step length . TherefoOptoelectronic systems are the gold standard for motion analysis in sports and rehabilitation ,14 and cThe present study focuses on step length during maximal sprint running. In a recent study, step length (and joint kinematics) during sprinting was recorded with an optoelectronic set up consisting of 21 cameras . Fortuna−1) and constant velocities ). Predicted 60-m time (7.59 ± 0.49 s) of the feet also closely matched the corrected 60-m gate time  −0.0025 s, LOA [−0.0056 0.00058]. Thus, fitting a mono-exponential velocity-time function solely based upon the measured 30-m (split) and 60-m times (−0.045 s) introduced systematic errors of only a few milliseconds. Thus, during maximal sprint acceleration, mono-exponential velocity-time functions constructed with the 30-m (split) and 60-m (finish) times as input accurately describe the increase of step velocity during individual sprint accelerations.The predicted 30-m split time of the feet obtained with the timing gate based exponential velocity-time functions was 4.28 ± 0.25 s. It closely matched the corrected gate time at 30-m  and similar to the measured video steps and strides  and similar to measured reference step-length . Based on the interpolation of these steps, the feet, on average, crossed the 30-m and 60-m lines 0.045 s earlier than the measured gate times. Thus, the tip of one of the feet crossed the 30-m and 60-m lines slightly ahead of the chest, while the chest usually contacted the mechanical timing rods. At the 30-m line, the average velocity was about 8.5 m·s−1 for the current athletes, which means that the tip of one of the feet was ~0.38 m (=8.5 m·s−1 × 0.045 s) ahead of the chest at ground contact. This distance is in line with the touchdown distance (about 0.32 m) reported for physical education students running at 8.04 m·s−1 [Horizontal velocity increases mono-exponentially during sprint acceleration  [−1 in their analysis and showed that the error of stride length estimations increased above 5 m·s−1 for the different algorithms that they evaluated [−1 already after two or three steps, and attained maximal velocities ranging from 8.0 to 10.5 m·s−1. Despite the inclusion of the acceleration phase and these high speeds, the errors in step length estimation were similar compared to the studies of Brahms et al. [To the best of our knowledge, our estimations of step length are considerably more accurate than previous predictions at high running speeds. Deriving accurate estimates of step length during maximal sprint acceleration from the IMU-signals using sensor fusion techniques ,24 is no−1) . Zrenner−1)  and the valuated . The aths et al.  and Zrens et al. . TherefoThe runners’ chest did not always contact first with the rods of our mechanical timing gates. Occasionally the arm or even the hand contacted the rod, which introduced some variation in the sprint times, but this also is the case with single beam optical timing gates.The manual digitization of videos introduced a random error of a few cm in our reference step lengths. Unfortunately, we did not have 40 m of a more accurate optical measurement system at our disposal that others used to obtain step length ,7.Our unsmoothed estimates of step length were subject to some error  sprints are commonly used to evaluate sprint performance in many sports. In principle, the velocity-time function of sprinters can also be derived with a series of more than two  timing gates or directly with radar or laser equipment. Although the latter devices measure instantaneous velocity rather well, the data need filtering and the accuracy depends on properly aiming these devices at the sprinters lower back . In pracThe athletes in the present study did not use starting blocks, because block-starts require a technique that was not mastered to the same extent by all the athletes. Football, rugby, and soccer players also do not use starting blocks, which facilitates the generalization of the present results to other sports. An additional strength of the present study is that the performance of the present athletes covered quite a broad range, although it unfortunately only included three women. Thus, our results are representative of athletes with different sprint capacities. The foregoing considerations suggest that the proposed method of step length estimation most likely applies to sportsmen and women in many other sports.Speed is the product of step rate and step length. IMUs attached to the shoes of athletes provide valuable information about ground contact time, airtime and step rate, which together with step length are key performance determining variables in sprinting ,3,5,6,7.In conclusion, 30-m and 60-m times in combination with IMU-derived step timing suffice to obtain good estimates of stride and step length during maximal sprint acceleration. Step length estimates complement other important sprint performance related variables  that can be accurately measured with IMUs and are used by coaches to monitor the training process and technical development of their athletes.